Unknown,Transcriptomics,Genomics,Proteomics

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HITS-CLIP analysis of FMRP mRNA binding sites from P11-P25 mouse brain polysomes

ABSTRACT: FMRP loss-of-function causes Fragile X Syndrome (FXS) and autistic features. FMRP is a polyribosome-associated neuronal RNA-binding protein, suggesting that it plays a key role in regulating neuronal translation, but there has been little consensus regarding either its RNA targets or mechanism of action. Here we use high throughput sequencing of RNAs isolated by crosslinking immunoprecipitation (HITS-CLIP) to identify FMRP interactions with mouse brain polyribosomal mRNAs. FMRP interacts with the coding region of transcripts encoding pre- and postsynaptic proteins, and transcripts implicated in autism spectrum disorders Polyribosomes were prepared from UV254-crosslinked pooled littermate FVB mouse brains as described in detail in published paper PMID 21784246. Polyribosomes were dissociated under denaturing conditions using two different protocols to disrupt ribonucleoprotein complexes. In most experiments Fmr1-null littermates were used in parallel to confirm FMRP specificity. After mild RNAse treatment to reduce the size of RNA to 60-100 nucleotides, endogenous FMRP was immunoprecipitated with one of two antibody combinations, either mixed monoclonal antibodies 7G1-1 and 2F5, or polyclonal antibody ab17722 (Abcam). The ages of the mice were P11, P13, P14, P15, and P25. In addition, to assess a different neuronal polysome-associated protein, as another control, lysates prepared from two samples, the P11 and P13 mice, were split in half and one half IPed with a human anti-Hu antisera as the neuronal Hu proteins are polysome-associated in brain. In sum, 7 FMRP HITS-CLIP experiments were performed including (1) P14 mouse polysomes prepared by Protocol 1 and IPed with 7G1-1/2F5 (2) P14 mouse polysomes prepared by Protocol 1 and IPed with ab17722 (3) P15 mouse polysomes prepared by Protocol 1 and IPed with 7G1-1/2F5 (4) P25 mouse polysomes prepared by Protocol 1 and IPed with 7G1-1/2F5 (5) P25 mouse polysomes prepared by Protocol 1 and IPed with ab17722 (6) P11 mouse polysomes prepared by Protocol 2 and IPed with ab17722 and (7) P13 mouse polysomes prepared by Protocol 2 and IPed with ab17722. FMRP-bound or Hu-bound RNA tags were cloned and submitted for high throughput sequencing on the Life Science 454 platform (samples 1-5) or the Illumina platform (samples 6 and 7 and the 2 Hu samples processed in parallel with samples 6 and 7). Full details are given in PMID 21784246.

ORGANISM(S): Mus musculus

SUBMITTER: Jennifer Darnell

PROVIDER: E-GEOD-45148 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Topoisomerases are crucial to solve DNA topological problems, but they have not been linked to RNA metabolism. Here we show that human topoisomerase 3M-NM-2 (Top3M-NM-2) is an RNA topoisomerase that biochemically and genetically interacts with FMRP, a protein deficient in Fragile X syndrome and known to regulate translation of mRNAs important for neuronal function and autism. Notably, the FMRP-Top3M-NM-2 interaction is abolished by a disease-associated FMRP mutation, and several human genetic studies link Top3M-NM-2 mutation to schizophrenia and intellectual disability. Top3M-NM-2 binds multiple mRNAs encoded by genes with neuronal functions related to schizophrenia and autism. Expression of one such gene, ptk2/FAK, is reduced in neuromuscular junctions of Top3M-NM-2 mutant flies. Synapse formation is defective in Top3M-NM-2 mutant flies and mice, as observed in FMRP mutant animals. Our findings suggest that Top3M-NM-2 acts as an RNA topoisomerase and works with FMRP to promote expression of mRNAs critical for neurodevelopment and mental health. We have identified Top3M-NM-2 as the first RNA topoisomerase in eukaryotes. To study whether Top3M-NM-2 binds mRNAs in vivo and also to identify its genome-wide RNA targets, we performed HITS-CLIP (high-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation; Licatalosi et al. Nature 456, 464-469, 2008). In this method, proteins are covalently crosslinked with bound RNAs in vivo, thus allowing specific mapping of the sites of interaction of the protein with the RNA. We cultured a HeLa cells stably-expressing HF-Top3M-NM-2 and irradiated the cells with UV to crosslink RNA and proteins in vivo. Following cell lysis, RNA was partially digested using various concentrations of RNase A. The RNA crosslinked to HF-Top3M-NM-2 was then immunoprecipitated with anti-Flag M2 argarose beads (Sigma). As a control, we also performed a mock immunoprecipitation using HeLa cells that do not express HF-Top3M-NM-2. The 3M-bM-^@M-^Y ends of the purified RNAs were ligated to a p32-labeled RNA adapter, fractionated by SDS-PAGE, and transferred to a nitrocellulose membrane. The p32-labeled RNA-Top3M-NM-2 complexes were detected by autoradiography and recovered from the membrane. The complexes were treated with Proteinase K to remove proteins. The RNA was ligated at their 5M-bM-^@M-^Yend to another RNA adaptor, and reverse-transcribed to cDNA. The cDNA obtained was fractionated a denaturing 6% TBE-Urea gel (InVitrogen), amplified by PCR, and subjected to high-throughput sequencing by Genome Analyzer II (Illumina). Sequenced tags were mapped to the human genome (hg18, NCBI/NIH) and to human RefSeq genes using ELAND program (Illumina).

Project description:Protein-RNA interactions are integral components of nearly every aspect of biology including regulation of gene expression, assembly of cellular architectures, and pathogenesis of human diseases. However, studies in the past few decades have only uncovered a small fraction of the vast landscape of the protein-RNA interactome in any organism, and even less is known about the dynamics of protein-RNA interactions under changing developmental and environmental conditions. Here, we describe the gPAR-CLIP (global photoactivatable-ribonucleoside-enhanced crosslinking and immunopurification) approach for capturing regions of the transcriptome bound by RNA-binding proteins (RBPs) in budding yeast. We report over 13,000 RBP crosslinking sites in untranslated regions (UTR) covering 72% of protein-coding transcripts encoded in the genome, confirming 3M-bM-^@M-^Y UTRs as major sites for RBP interaction. Comparative genomic analyses reveal that RBP crosslinking sites are highly conserved, and RNA folding predictions indicate that secondary structural elements are constrained by protein binding and may serve as generalizable modes of RNA recognition. Finally, 38% of 3M-bM-^@M-^Y UTR crosslinking sites show changes in RBP occupancy upon glucose or nitrogen deprivation, with major impacts on metabolic pathways as well as mitochondrial and ribosomal gene expression. Our study offers an unprecedented view of the pervasiveness and dynamics of protein-RNA interactions in vivo. Duplicate gPAR-CLIP and mRNA-seq libraries were sequenced from yeast strains for each of three conditions: log-phase growth, growth after 2 hour glucose starvation, and growth after 2 hour nitrogen starvation. Additional duplicate mRNA-seq libraries were sequenced from yeast strains grown in the absence of 4-thiouracil. gPAR-CLIP libraries were used to determine regions of mRNA bound by proteins. mRNA-seq libraries served as controls for mRNA abundance. A Puf3p PAR-CLIP library was sequenced to determine how well gPAR-CLIP captured the binding signatures of a single RNA-binding protein.

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HITS-CLIP analysis of FMRP mRNA binding sites from P11-P25 mouse brain polysomes

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